A crucial element in the development of effective therapeutic and prophylactic strategies for AIDS is an experimental animal model in which the course of immunodeficiency virus infection parallels the pathogenesis of the human disease. The SIV infection of macaques is such a model since this virus induces an immunodeficiency syndrome in infected macaques that is remarkably similar to human AIDS. Therefore, candidate vaccines can be evaluated not only for their ability to prevent infection but also for their ability to prevent AIDS. The major vaccine effort within the laboratory has been an evaluation of the highly attenuated vaccinia virus Ankara (MVA) strain as a recombinant vector used for priming that is followed by a subunit SIV boost. Interest in this approach is based upon a pilot study of 12 macaques in which animals immunized with the MVA-SIV recombinant exhibited significant down modulation of virus replication that was associated with prolonged survival. Two of these vaccinated macaques have remained healthy for 4 years. Modulation of viremia appeared to correlate with a gag-specific antibody response observed prior to SIV challenge. These studies demonstrated that MVA was as effective as conventional vaccinia viruses such as the New York Board of Health in eliciting an immune response in monkeys. In addition, this is the first study to demonstrate that immunization can modulate viremia and disease progression in the absence of sterilizing immunity in the SIV/ macaque model for AIDS. We wished to explore MVA as a live virus vaccine in the SIV model with the eventual goal of optimizing this approach for future development as a potential AIDS vaccine for humans. The short- term goals of this project are twofold: to define the antigens required to mediate protection and to investigate the role of gag-specific CTL in this effect. Three second generation recombinant MVA viruses were generated that expressed either gag- pol alone, env alone, or a combination of gag-pol and env, with all genes being expressed from the strong early/late synthetic promoter. A cohort of 24 rhesus macaques have been immunized (six per immunogen) to evaluate immunogenicity with the eventual goal of analyzing the kinetics of viremia in these animals after intravenous challenge and attempt to correlate any effects on viral load with immunogen. Four sequential immunizations resulted in moderate boosting of both gag and env-specific antibody responses. These macaques were challenged with cell free SIVsmE660 and as expected all of the macaques became infected; however, as of eight weeks after challenge, significant differences in CD4 lymphocyte numbers between the control monkeys and those immunized with SIV antigens in the peripheral blood are emerging. These animals are presently being evaluated for plasma and lymphoid virus load. Evaluation of gag-specific CTL in vaccinia-immunized macaques presents a considerable challenge and therefore in collaboration with Dr. Norman Letvin, utilized macaques expressing the MamuA*01 MHC Class I haplotype for which a dominant gag-specific epitope (p11 C) has been identified. In order to select MamuA*01 macaques, we developed PCR primers, and conditions to screen 68 macaques and identified six animals which expressed this haplotype. Four of these macaques were immunized with the MVA- gag-pol recombinant virus at 0 and 13 weeks and an additional two macaques were immunized with MVA and sequential generation of a p11C-specific CTL response was measured utilizing both traditional CTL assays as well as MHC Class I/Peptide tretramer staining. Macaques immunized with MVA expressing gag-pol developed CD8+ CTL as early as 2 weeks after the first immunization (2 of 4) which was boosted in all animals to high levels after the second immunization. CTL could be demonstrated in 1 to 3% of peripheral blood lymphocytes and lymph nodes by MHC Class I/Peptide tretramer staining after the second immunization. These levels are equivalent to those observed in SIV-infected macaques suggesting that this vaccine strategy approaches the cell mediated immune response achieved in such approaches as attenuated live virus vaccines.